Flagellar dynein ATPases are large, multi-subunit motor enzymes. Most cilia and flagella have two non-identical rows of dyneins attached to each doublet microtubule, both of which contribute to microtubule sliding and flagellar motility. Dyneins of the outer row more closely resemble cytoplasmic dyneins in containing two or three ca. 500 kd catalytic heavy chains, two Ca. 75 kd intermediate chains and multiple light chains. While the role of dyneins as microtubule-associated motors for both ciliary and cytoplasmic motility is well recognized, little is known about their regulation or their mode of attachment to the loads they carry (vesicles in the cytoplasm, doublet microtubules in cilia and flagella). Long term goals of the proposed experiments are to determine the roles of flagellar dynein heavy chains, intermediate chains and light chains in flagellar motility, and to characterize interactions between dynein subunits and doublet microtubule-associated proteins that are important for dynein attachment during flagellar assembly and for regulation of outer row dynein motors. Using the outer row dynein of chlamydomonas reinhardtii flagella as a model system, genes essential for normal dynein assembly and function are being cloned, sequenced, and genetically mapped. The function of each subunit in the generation and regulation of dynein motor activity is then being analyzed by phenotypic characterization of mutations at each locus and by in vitro alteration and in vivo expression of cloned genes. Experiments proposed here are designed to explore structure-function relationships in the alpha and beta heavy. chains and 70 kd intermediate chain, and to use insertional inactivation mutagenesis to clone additional dynein assembly genes.